CN107525923A - One kind centrifuges immunochromatography detection method and device - Google Patents
One kind centrifuges immunochromatography detection method and device Download PDFInfo
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- CN107525923A CN107525923A CN201710212197.9A CN201710212197A CN107525923A CN 107525923 A CN107525923 A CN 107525923A CN 201710212197 A CN201710212197 A CN 201710212197A CN 107525923 A CN107525923 A CN 107525923A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6887—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids from muscle, cartilage or connective tissue
Abstract
The invention discloses the present invention to provide a kind of centrifugation immunochromatography detection method, methods described is characterized by centrifugal device driving liquid phase is used on the basis of existing immunochromatography technique in flowing during solid phase detects film, and detection method is chromatographed available for colloidal metal immunochromatography detection method, fluorescence immune chromatography detection method and chemiluminescence immunoassay.The present invention has high sensitivity, detection time is short, easy to use, stability is high, is easy to the characteristics of storage.
Description
Technical field
The present invention relates to one kind to centrifuge detection method and device, belongs to technical field of immunoassay.
Background technology
Immunology detection technology is measure antigen, antibody, immunocyte and the chemical composition of applied immunology principle design
Deng laboratory facilities, the sample of medical diagnosis on disease and health detection can be carried out from human body and animal body and be used for by being widely used in
Environment, Pharmaceutical Analysis, food and the sample of Industrial Analysis.Conventional has immune turbidity technology, solid-phase enzyme immunoassay technology, changes
Learn luminescent detection techniques, immunofluorescence label technology, flow cytometry, colloidal gold technique etc..Immune turbidity technology, it is also referred to as immune
Nephelometry is soluble antigen, antibody specific bond in the liquid phase, produces a certain size compound, forms the refraction or suction of light
Receive, determine the transmitted light after this refraction or absorption or scattering light as unit of account, for quantitatively detecting, but detection sensitivity
It is low, it is not suitable for trace detection.The enzyme of immobilization and antigen or antibody of the solid-phase enzyme immunoassay technology based on antigen or antibody
Mark, is incorporated in the antigen of surface of solid phase carriers or antibody keeps its immunologic competence, and the enzyme conjugates of antigen or antibody was both protected
Its immunologic competence is stayed, retains the activity of enzyme again, in measure, by inspection sample（Determine antibody or antigen therein）With enzyme mark
Antigen or antibody react by the antigen or antibody of different step and surface of solid phase carriers, have high sensitivity, linear response
The remarkable advantages such as scope is wide and easy to automate, but detect reaction time length and limit its use.Immunochemiluminescence is examined
Survey technology is a kind of highly sensitive micro and Analytical Methods of Trace, have easy to operate, high sensitivity, linear response range wide and
It is easy to automate to wait remarkable advantage, it is widely used in environment, clinic, Pharmaceutical Analysis, food and Industrial Analysis, and
The luminescence reagent labelling technique of solid phase separation means and antigen or antibody based on antigen or antibody, but detection reaction time length,
Detection reagent needs stored refrigerated and transport and the requirement height on detection device to influence its use.Immunofluorescence label technology, stream
Formula cell art, colloidal gold technique are also that conventional detection technique is widely used, but have its corresponding deficiency, when detection is reacted
Between long or sensitivity, Stability and veracity shortcoming be generally existing deficiency.It is high sensitivity, quick, convenient, miniaturization, complete
Quantitative, automation is the development trend of current clinical immunization detection technique product, but existing can not all realize above-mentioned work(simultaneously
Energy.
The content of the invention
It is an object of the invention to provide one kind to centrifuge immunochromatography detection method and device.The present invention has sensitivity
Height, detection time is short, easy to use, stability is high, is easy to the characteristics of storage.
The present invention provides a kind of centrifugation immunochromatography detection method, and methods described is with using centrifugal device driving liquid
Mutually flowed in immunochromatography solid phase detects film, wherein the immunochromatography includes the colloidal metal using colloidal metal as indicator
Immunochromatography, fluorescence immune chromatography and chemiluminescent substance and/or chemiluminescence enzyme using fluorescein as indicator mediate luminous
At least one that chemiluminescence immunoassay as indicator chromatographs.
In above-mentioned method, the colloidal metal is at least one of collaurum, electroselenium and colloid gold-magnetic particles；Institute
Stating fluorescein includes fluorescein isothiocynate, RB 200, TRITC, phycoerythrin, more dinoflagellates
Flavine phyllochlorin, propidium iodide, other at least one of phycocyanin and europium compound；The chemiluminescent substance includes
Luminol and different luminol and its derivative species, acridinium ester and a word used for translation shallow lake amide-type, (golden steel alkane) -1,2- dichloroethanes and its derivative
At least one of thing and tris (bipyridine) ruthenium；The chemiluminescence enzyme includes HRPO, alkaline phosphatase and xanthine
At least one of oxidizing ferment.
The chemical luminous substrate of the HRPO often uses luminol and different luminol and its derivative species, such as different Shandong
The different Shandong promise of base-N- ethyls of minot, 4- amino and AHEI and ABEI etc., currently used product have the production of PIERCE companies
West Pico chemiluminescence detections substrate, West Dura chemiluminescence detections substrate, West Femto chemiluminescence detections
Substrate.The chemical luminous substrate of alkaline phosphatase it is conventional have (golden steel alkane) -1,2- dichloroethanes and its derivative, AMPPD,
CDP-STAR、Lumi-Phos 530.The chemical luminous substrate of xanthine oxidase has xanthine, myricetin, Quercetin.
In above-mentioned immunochromatography chemical luminescence detection method, in addition to non-enzyme-catalyzed chemical luminescence substrate, i.e., it is directly chemical
Luminescent substance, it is the immunoassay method with the direct labelled antigen of chemiluminescence agent or antibody.It is usually used in the chemiluminescence of mark
Material has acridine ester type compound-acridinium ester (AE), is effective luminous marker, and it is sent out by starting
Light reagent（NaOH、H2O2）Act on and light, mainly there is acridinium ester and a word used for translation shallow lake amide-type, tris (bipyridine) ruthenium etc..
In above-mentioned method, the solid phase detection film includes nitrocellulose filter, pvdf membrane, polyvinylidene fluoride film, nylon
Film, one kind of DEAE cellulose membranes or combination.
In above-mentioned method, methods described is the carrying carrier using particulate as the indicator；The carrying carrier is held
The mode for carrying the indicator is used with the thing specificity to be checked that the indicator directly marks directly in conjunction with thing or thing to be checked spy
Different in nature indirect conjugates mark the particulate or directly mark thing specificity to be checked straight with the particulate with the indicator
Binding compound or thing specificity indirect conjugates to be checked；The particulate is directly and/or can pass through the mode of being chemically crosslinked and egg
White matter and/or the indicator form non-specific binding and maintain stable particle, the particle diameter selection 1nm- of the particulate
1um, concretely 10 ~ 800 nm, 20 ~ 600 nm or 43 ~ 500 nm；The specificity junction mixture includes antigen, antibody, parent
With element, biotin and its derivative.Currently used particulate has colloidal gold particles, colloidal selenium particles, colloid gold-magnetic particles, fluorescence
Microballoon, magnetic particle, gold-magnetic particles, microgel particle, emulsion particle, plastic microsphere, microsphere silica gel, agarose beads, polyphenyl second
Alkene particulate, silicon dioxide microsphere, polystyrene microsphere, carboxyl microballoon, chloromethyl microballoon etc..
In above-mentioned method, the liquid phase includes the liquid phase containing thing to be checked, with the detectable substance of the tracer-labelling
Liquid phase, the liquid phase of the detectable substance marked with the particulate, the liquid phase of the detectable substance of non-marked, the one kind for cleaning liquid phase or combination；
The detectable substance is that thing specificity junction mixture to be checked and its two level or three-level specificity junction mixture, the specificity junction mixture include
Antigen, antibody, Avidin, biotin and its derivative.
In above-mentioned method, methods described is to contain ox blood also containing solid phase detection membrane closure liquid, the confining liquid
Pure albumen and other soluble proteins, skimmed milk power, polyethylene glycol, casein, sucrose, surfactant, gelatin, blood
Clearly, the buffer salt solution of at least one of blood plasma, including step used below：
1）Film coating buffer is detected using solid phase and is coated with the solid phase detection film, the solid phase detection film coating buffer includes antigen, resisted
Body, Avidin, the solution of biotin and its derivative；
2）The solid phase detection film is cleaned using the cleaning fluid, the cleaning fluid includes water, conventional buffers and its contains table
The cushioning liquid of face activating agent；
3）Oozed with confining liquid leaching and cover the solid phase detection film；
4）The solid phase detection film is cleaned again with the cleaning fluid；
5）Dry, it is standby.
One kind, which centrifuges detection means, includes sample introduction part and centrifugal device；The centrifugal device is included by motor
The centrifugal rotor and support base of driving, the centrifugal rotor is using the support base as support；The sample introduction part not with from
Heart rotor is directly connected to, located at the top of centrifugal rotor, lower section or outside；The sample introduction part includes liquid phase storage device, entered
Sample pipe and sampling pump；The liquid phase storage device is connected with the sample feeding pipe；The sampling pump drives the liquid phase storage dress
Liquid in putting enters the sample feeding pipe；The sample feeding pipe directly or indirectly loads the liquid phase to the solid phase and detects the near of film
On heart side；The solid phase detection film, which is placed on the centrifugal rotor, to be centrifuged.
In above-mentioned device, the immobilon-p is placed in immobilon-p fixing device, and the immobilon-p fixing device is selected from solid
At least one of phase film support egative film sample part, lateral flow test strips buckle sample part and hyalomitome embedding formula part, and it is described
Immobilon-p fixing device is placed on the centrifugal rotor；The immobilon-p fixing device is dismountable with the centrifugal rotor
Structure.
In above-mentioned device, described device contains detector, including collaurum quantitative detector, fluorescence detector, chemistry
One kind of luminescence detector or combination, the Testing index of the detector include absorbance, fluorescent value, values of chemiluminescence and image
Any of digital signal value or combination.
In above-mentioned device, the sample introduction speed of the rotating speed of the centrifugal device and the sampling device uses programme-control
Mode；The rotating speed of the centrifugal device is 200 ~ 10000 r/min, concretely 500 ~ 5000 revs/min, 800 ~ 3000
Rev/min or 800 ~ 2000 revs/min.
Method any one of above-mentioned method or apparatus or described device are in immunologic detection method product development
Using.
For the present invention due to taking above technical scheme, it has advantages below：
1st, the present invention improves thing to be checked using liquid phase flowing and cleaning on solid phase detection film of centrifugal device driving detection
Capture binding ability, reduce solid phase detection film ambient noise interference, improve method detection sensitivity, realize with existing
The high-sensitivity detection of detection reagent.
2nd, the present invention is flowed using the liquid phase of centrifugal device driving detection on solid phase detection film, changes existing film inspection
The present situation that survey method flows by nature and liquid reduces with the extension of flow on film, can keep liquid even on film
Speed flowing, ensure that the homogeneity that thing to be checked combines on film, can improve detection accuracy.
3rd, existing film detection method flows by nature, and flowing velocity of the liquid on film slows down over time, completes
One detection generally requires the time of more than 15 minutes.And the present invention detects film using centrifugal device driving detection liquid phase in solid phase
Upper flowing, maintain liquid and at the uniform velocity flowed on film, it is quick to shorten detection time.
4th, existing high-sensitivity detecting method, using multi-step, too many levels drive control, it is related to detection sample, inspection
Survey the transposition and movement of phase and reaction carriers.And the present invention is using centrifugal device driving liquid phase flowing and sampling pump sample introduction, behaviour
It is simple to make step.
5th, met directly in existing film detection method using tracer-labelling detection and produce very strong background signal, can not
Carry out the detection of thing to be checked.Inventive closure fluid-tight, which is closed, can substantially reduce background signal, and eccentric cleaning can be removed effectively
Free the tracer-labelling thing and indicator remained in background, effectively improves detection efficiency and accuracy.
6th, operating procedure of the present invention is simple, it is easy to accomplish automation mechanized operation.The inventive method has high sensitivity, Quan Ding
The characteristics of amount, automation, while have that detection is quick, uses the simple detection method of equipment again；Not only easy to use, reduction original
The waste of material, while operating efficiency is also significantly improved, applied to detection and analysis, the numerous areas of separation.
Brief description of the drawings
Fig. 1 is the schematic diagram that the present invention centrifuges detection means.
Fig. 2 is that immobilon-p supports egative film sample modular construction schematic diagram.
Fig. 3 is the fixer structure schematic diagram of immobilon-p.
Fig. 4 is immobilon-p and sample introduction component locations structural representation.
Fig. 5 is the structural representation that immobilon-p fixator hyalomitome embeds formula part up and down.
Fig. 6 is immobilon-p and ultrasonication device positional structure schematic diagram.
Embodiment
The present invention is further described following embodiment combination accompanying drawings, but the invention is not limited in following implementation
Example.
Embodiment 1, horizontal type centrifuge the preparation of detection means
Include as shown in figure 1, the present invention centrifuges detection means：Sample introduction part 1, solid phase detection film 2, centrifugal device（Centrifugation
Rotor 3；Motor 5；Support base 6）With detector 4.Include ultrasonication device 7 and shell 8 simultaneously.Sample introduction part 1 is right
Answer solid phase detection film 2 proximal part, ultrasonication device 7 correspond to solid phase detection film 2 detection position, centrifugal device have from
Heart rotor 3, motor 5 and support base 6 form, and motor 5 is located in support base 6, and centrifugal rotor 3 is by motor
5 driving rotations.
As depicted in figs. 1 and 2, solid phase detection film 2 is provided with the proximal part of centrifugal rotor 3 and is attached thereto logical liquid adsorption
Discrete part 9, solid phase detect distal end of the film 2 in centrifugal rotor and examined provided with logical liquid collecting member 10, solid phase is attached thereto
The fixed placement of film 2 is surveyed in the outer of centrifugal rotor 3, is dismountable structure with centrifugal rotor 3, liquid adsorption discrete part 9 is
The liquid phase sample loading position of sample introduction part.Wherein, the material of solid phase detection film 2 uses nitrocellulose filter, pvdf membrane, gathered partially
Any of fluoride film, nylon membrane and DEAE cellulose membranes, and the one or both sides of solid phase detection film 2 are provided with backing；Liquid
Body absorption discrete part 9 includes colloid gold label adsorbed film, fluorescent labeled antibody adsorbed film, chemiluminescent labeling adsorbed film and divided
Dissipate at least one of film；Fluid collection device 10 uses water-absorbing material, such as blotting paper and/or water absorbent gel, it is also possible to liquid
Body collection vessel.
As shown in Figure 2 and Figure 3, a kind of solid phase for the setting of film 2 being detected for fixed solid phase detects film fixing device, as propped up in Fig. 2
Egative film 11 is supportted, PVC board, transparent plastic sheet, poly (methyl methacrylate) plate etc. may be selected in support egative film 11.Solid phase detection film fixes dress in Fig. 3
Lateral flow test strips buckle sample part 12 is set to, specifically includes poroid part 13, point sample groove 14, observation window 15 and liquid collection portion
Part outlet 16.After solid phase detection membrane structure is put into lateral flow test strips buckle spline structure 12, the corresponding position of point sample groove 14 is liquid
Body adsorbs discrete part 9, and the corresponding position of observation window 15 detects film 2 for solid phase, and liquid collecting member exports 16 corresponding positions and is
Liquid collecting member 10（For water-absorbing material or liquid collecting container）.
As shown in Figure 1 and Figure 4, sample introduction part 1 includes closing phase storage device 17, liquid phase storage device 18, sampling pump 19
With sample feeding pipe 20.Closing phase storage device 17 and liquid phase storage device 18 are connected with sample feeding pipe 20, located at the upper of centrifugal rotor 3
Side, and liquid phase 21 is driven into sample feeding pipe 20 by sampling pump 19, then adds to point sample groove 14.In order to control the speed of centrifugal device
With the sample introduction speed of sampling device, centrifugal device and sample introduction part are connected with the part with programme-control speed.
As shown in figure 5, another solid phase that the setting of film 2 is detected for fixed solid phase detects film fixing device, specifically include hard
The transparent lower cover slip 22 of matter, hard transparent upper cover plate 24, preceding naked empty interlayer 23 and rear naked empty interlayer or liquid collecting member 10.Detection
When liquid phase by centrifugation, premenstrual naked empty interlayer 23, into liquid adsorption discrete part 9, flow through solid phase detection film 2, it is reacted
Liquid phase is discharged from rear naked empty interlayer or liquid collecting member 10.
As shown in figures 1 to 6, ultrasonication device 7 includes ultrasonic generator 25, transducer 26, ultrasonic transformer 27 and ultrasound
Broken container 28, positioned at the top of solid phase detection film 2, in use, ultrasonic transformer 27 declines, the detection zone of cutting solid phase detection film 2
Domain, it is pushed into ultrasonication container 28, ultrasonication, then device detects after testing.In order to control cutting and broken controllability
With uniformity, centrifugal device, sample introduction part, ultrasonication device and detector are connected with the part with programme-control speed
Connect.
The experimental study of the present invention：The detection method and its effect of following description of test present invention, but be not to the present invention
Restriction.Experimental method used in following experiments is conventional method unless otherwise specified.Material used in following experiments
Material, reagent etc., unless otherwise specified, commercially obtain.
Experiment one, the comparison of the detection correlation of the invention with existing detection method
First, experiment material
Anti-human myoglobins polyclonal antibody（Genagates companies of the U.S.）, anti-human myoglobins monoclonal antibody（The U.S.
Genagates companies）, spectrophotometer（Shanghai mountain valley with clumps of trees and bamboo China Tech skill Instrument Ltd., 752 ultraviolet-uisible spectrophotometers）, people
Myoglobins（Sigma-Aldrich products）, BioFlow die instrument（IMAGENE companies of the U.S.）, Index cutting machines（It is beautiful
A-point companies of state）, DBF -900 sealing machines（Wenzhou south of the River packing factory）, ACBO dehumidifiers（Jiangsu wuxi Austria ripple dehumidifier
Company）, desk centrifuge（Eppendoff companies of the U.S.）, bovine serum albumin(BSA)（Abbreviation BSA, SIGMA product）, cellulose nitrate
Plain diaphragm（AE 99, provided by Genagates companies of the U.S.）, multi-polyester cellulose membrane (Reemay 2033, the U.S.
Alstrom Products), absorb water paper membrane pad（Grade 470, U.S.'s S＆S Products）, gold chloride（SIGMA products）, colloid
The quantitative chromatographic analysis instrument of gold（Norway's Skannex products）.
2nd, experimental method
The preparation of human muscle hemoglobin solution：The human muscle hemoglobin solution of concentration known is taken, uses sample dilution buffer（1%BSA,
100mM glycine, 50mM PBS, 150mM NaCl, pH7.4）Dilution configuration 3.125,6.25,12.5,25,50,100ng/
Ml serial human muscle hemoglobin solution.
The preparation of the anti-human myoglobins monoclonal antibody of colloid gold label：10ml pure water is taken, heating stirring, treats boiling water
When add the chlorauric acid solutions of 500 μ l 10%, heating is boiled 5 minutes, is added the citric acid three sodium solutions of 500 μ l 12%, is kept this molten
Liquid stirring boiling 10 minutes, is naturally cooling to room temperature, i.e. colloidal gold solution.Colloidal gold solution volume 10ml is taken, with 10% potassium carbonate
PH to 8.3 is adjusted, is rapidly added the anti-human μ g of myoglobins monoclonal antibody 100, to 10 μ g/ml final concentrations, rocks beaker mixing, room
Temperature is placed 30 minutes, is rapidly added 10% bovine serum albumin solution 100ul, is made final concentration of 1%, while shakes beaker, room temperature
Place 30 minutes, 12000rpm is centrifuged 20 minutes, carefully suctions out supernatant；5ml 50mM phosphate (PBS) buffer solution is added,
PH7.4, precipitation is suspended, 12000rpm is centrifuged 20 minutes, suctions out supernatant, is dissolved in the cow's serum that 1.0ml contains 1% by precipitating
In the phosphate buffer of albumin and 3% sucrose, 4 DEG C are kept in dark place.
Colloid gold label adsorbs film preparation：Preparation contains 0.5%PVA（That is polyvinyl alcohol）, 50mM PBS liquid, 0.5%BSA,
0.88% NaCl, pH 7.4 multi-polyester cellulose membrane pretreatment fluid, pending multi-polyester cellulose membrane is put in pretreatment fluid
It is interior, soaking at room temperature 1 hour, take the film out, put and seal after 37 DEG C of dryings standby, can also be used directly as dispersion membrane.Take colloid
Golden labelled antibody solution, with colloid buffer solution（1%BSA, 3% sucrose, 50mM PBS, pH7.4）It is 30 to be diluted to OD530, is started
Die instrument, load antibody, take multi-polyester cellulose membrane, start die, set die condition as：The airbrush translational speed 30mm/ seconds,
The μ l/cm of liquid fltting speed 3.0, the film after printing is put into drying box, 37 DEG C of dryings 6 hours, is subsequently placed in containing drier
Hermetic bag in preserve use.
It is prepared by polyclonal antibody die：Anti-human myoglobins Anti-TNF-α liquid solution is taken, with 50mM phosphate buffers
(pH 7.4) is diluted to 1mg/ml concentration.Start die instrument, load antibody, take the PVC pieces for posting nitrocellulose filter（That is polychlorostyrene
Piece of vinyl）, start die, set die condition as：Airbrush translational speed 30mm/ seconds, the μ l/cm of liquid fltting speed 0.5.Will print
The film made is put into 37 DEG C of drying boxes, dries 6 hours, then film is placed in the drying receptacle containing drier and preserves use.
Semi-finished product assemble method：Starting dehumidifier makes the humidity in operating room be reduced to less than 25%, is printed in polyclonal antibody
Water suction paper membrane pad and colloid gold label adsorbed film are pasted in film both ends respectively, then with Pressure sensitive adhesive tape sealing label surface.Put what is pasted
Detection lug is on cutting machine, being cut into 3.5mm test strips.Paper slip is put into the aluminium amber hermetic bag of drier, on sealing machine
Sealing, labelling.
The test strips of above-mentioned preparation are taken, with the side of colloid gold label adsorbed film upward, are placed in centrifuge rotor（Extroversion is inclined
Oblique type）It is interior, to colloid gold label adsorbed film on be added dropwise preparation various concentrations human muscle hemoglobin solution 80ul, stand 1-15 point
Clock, 2000 revs/min centrifuge 1 minute, then the 50mM PBS 80ul to dropwise addition pH7.4 on colloid gold label adsorbed film,
2000 revs/min of centrifugations are cleaned for 1 minute, take out test strips, are positioned over the quantitative chromatographic analysis instrument of collaurum（That is detector）Upper reading
The digital picture of polyclonal antibody trace band, carry out image procossing and obtain corresponding chroma value.Control stripes bar do not do from
Heart processing, after the above-mentioned identical time of repose of setting, then stands 2.5 minutes, then reads corresponding chroma value.
Test in triplicate, results averaged.The detected value of statistical calculations difference pre-dwell time test strips.
3rd, experimental result
Detection method measurement result of the present invention using collaurum as indicator shows the correlation coefficient r of the technology of the present invention detection2It is flat
Average is 0.962, prior art detection（Do not do centrifugal treating）Correlation coefficient r2For 0.936, P ＜ 0.05, it is significantly better than existing
There is the testing result of technology, illustrate that the technology of the present invention improves the accuracy of prior art detection.Experimental result is as shown in table 1.
Testing result analysis of the accuracy of the present invention of table 1 using collaurum as indicator（Unit：Pattern colour angle value）
| Time（Point） | Technology | 3.125 | 6.25 | 12.5 | 25 | 50 | 100 | r2 |
| 1 | Current art | 19.8 | 19.7 | 23.0 | 28.8 | 31.4 | 43.7 | 0.928 |
| The present invention | 8.7 | 7.4 | 12.3 | 16.8 | 24.8 | 37.9 | 0.926 | |
| 2 | Current art | 20.3 | 21.2 | 23.3 | 27.6 | 38.6 | 44.0 | 0.932 |
| The present invention | 16.5 | 19.4 | 26.3 | 31.8 | 35.3 | 39.3 | 0.965 | |
| 5 | Current art | 34.1 | 32.1 | 38.0 | 42.3 | 49.1 | 62.6 | 0.902 |
| The present invention | 23.0 | 24.3 | 29.0 | 38.9 | 54.8 | 65.3 | 0.962 | |
| 10 | Current art | 32.3 | 31.4 | 40.1 | 45.9 | 61.7 | 73.8 | 0.947 |
| The present invention | 24.9 | 29.3 | 42.3 | 48.2 | 61.4 | 73.1 | 0.985 | |
| 15 | Current art | 33.2 | 33.5 | 43.1 | 54.3 | 64.7 | 76.7 | 0.972 |
| The present invention | 26.1 | 35.3 | 45.2 | 58.1 | 65.3 | 74.7 | 0.971 |
Experiment two, the of the invention and comparison of the limit of identification of existing detection method
First, experiment material
With experiment one
2nd, experimental method
With experiment one
3rd, experimental result
Measurement result of the present invention using collaurum as indicator, analysis are big using the conventional correlation coefficient r value of Related product exploitation
Requirement in 0.98 has carried out statistical disposition to data, and minimum value when being more than 0.98 with r values is set to limit of identification.As a result it is same
Under the experiment condition of sample, 2-10 minutes pre-dwell time, the limit of identification of prior art is used as 6.25ng/ml, using this
The limit of identification of invention is 3.125ng/ml, and detection sensitivity is significantly higher than prior art, illustrates that the technology of the present invention improves
The detection sensitivity of prior art detection.Experimental result is as shown in table 2.
Detection sensitivity of the present invention of table 2 using collaurum as indicator is analyzed（Unit：Pattern colour angle value）
| Time（Point） | Technology | 3.125 | 6.25 | 12.5 | 25 | 50 | 100 | r2 | Limit of identification |
| 1 | Current art | 19.7 | 23.0 | 28.8 | 31.4 | 43.7 | 0.970 | 6.25 | |
| The present invention | 7.4 | 12.3 | 16.8 | 24.8 | 37.9 | 0.995 | 6.25 | ||
| 2 | Current art | 21.2 | 23.3 | 27.6 | 38.6 | 44.0 | 0.963 | 6.25 | |
| The present invention | 16.5 | 19.4 | 26.3 | 31.8 | 35.3 | 39.3 | 0.966 | 3.125 | |
| 5 | Current art | 32.1 | 38.0 | 42.3 | 49.1 | 62.6 | 0.981 | 6.25 | |
| The present invention | 23.0 | 24.3 | 29.0 | 38.9 | 54.8 | 65.3 | 0.962 | 3.125 | |
| 10 | Current art | 31.4 | 40.1 | 45.9 | 61.7 | 73.8 | 0.991 | 6.25 | |
| The present invention | 24.9 | 29.3 | 42.3 | 48.2 | 61.4 | 73.1 | 0.986 | 3.125 | |
| 15 | Current art | 33.2 | 33.5 | 43.1 | 54.3 | 64.7 | 76.7 | 0.972 | 3.125 |
| The present invention | 26.1 | 35.3 | 45.2 | 58.1 | 65.3 | 74.7 | 0.971 | 3.125 |
Experiment three, the present invention are on detecting specific influence
First, experiment material
With experiment one
2nd, experimental method
With experiment one, human muscle hemoglobin solution 3.125,6.25,12.5,25,50,100ng/ml, same experiment condition are prepared
Under, 5 minutes pre-dwell time.
Make standard curve：Take concentration known human muscle hemoglobin solution 3.125,6.25,12.5,25,50,100ng/ml
Human muscle hemoglobin solution, of the invention and existing detection method is respectively adopted and detects and draws standard curve.
Specific detection specimen in use is A：50ng/ml myoglobins, B：10ng/ml Troponin Is, C：30ng/ml fleshes
Acid kinase isodynamic enzyme, D：80mg/ml human serum albumins, E：20mg/ml cholesterol.Prepared with sample dilution buffer.
3rd, experimental result
The present invention is using collaurum as the above-mentioned specific detection sample of the detection of indicator, and experimental result is as shown in table 3, current art
It is respectively 51 and 49ng/ml to repeat detection average value to myoglobins sample with the technology of the present invention, other not contain myoglobins
Sample detected value below the detection sensitivity lower limit of detection method, be feminine gender, and without obvious chromogenic reaction.Explanation
The present invention does not influence detection specificity.
The present invention of table 3 and the current art detection specific results contrast of myoglobins（Unit：ng/ml）
| Sample | Technology | Repeat 1 | Repeat 2 | Repeat 3 | Average value |
| A | Current art | 58 | 52 | 43 | 51 |
| The present invention | 55 | 48 | 44 | 49 | |
| B | Current art | ＜ 6.25 | ＜ 6.25 | ＜ 6.25 | ＜ 6.25 |
| The present invention | ＜ 3.125 | ＜ 3.125 | ＜ 3.125 | ＜ 3.125 | |
| C | Current art | ＜ 6.25 | ＜ 6.25 | ＜ 6.25 | ＜ 6.25 |
| The present invention | ＜ 3.125 | ＜ 3.125 | ＜ 3.125 | ＜ 3.125 | |
| D | Current art | ＜ 6.25 | ＜ 6.25 | ＜ 6.25 | ＜ 6.25 |
| The present invention | ＜ 3.125 | ＜ 3.125 | ＜ 3.125 | ＜ 3.125 | |
| E | Current art | ＜ 6.25 | ＜ 6.25 | ＜ 6.25 | ＜ 6.25 |
| The present invention | ＜ 3.125 | ＜ 3.125 | ＜ 3.125 | ＜ 3.125 |
Experiment four, the of the invention and comparison of the detection performance of existing chemical luminescence detection method
First, experiment material
Anti-human myoglobins polyclonal antibody（Genagates companies）, the anti-human myoglobins Dan Ke of horseradish peroxidase-labeled
Grand antibody（Genagates companies of the U.S.）, magnetic particle（MP-COOH-20020, Zhengzhou Ying Nuo bio tech ltd）、pico
Luminescence reagent（Thermo scientific）, chemiluminescence detector（Promega, Glomax Multi JR Detection
System）, human muscle hemoglobin（Sigma-Aldrich products）, BioFlow die instrument（IMAGENE companies of the U.S.）, Index
Cutting machine（A-point companies of the U.S.）, DBF-900 sealing machines（Wenzhou south of the River packing factory）, ACBO dehumidifiers（Jiangsu without
Xi Aobo dehumidifiers company）, desk centrifuge（Eppendoff companies of the U.S.）, bovine serum albumin(BSA)（SIGMA products）, nitric acid
Cellulose diaphragm（AE 99, provided by Genagates companies of the U.S.）, multi-polyester cellulose membrane (Reemay 2033, the U.S.
Alstrom Products), absorb water paper membrane pad（Grade 470, U.S.'s S＆S Products）.
2nd, experimental method
The preparation of human muscle hemoglobin solution：With experiment one.
1st, existing chemical luminescence detection method group
The mark of magnetic particle, using conventional labels method, magnetic particle is carried out with 1mg/ml anti-human myoglobins polyclonal antibodies
Mark, the amount ratio of anti-human myoglobins polyclonal antibody and magnetic particle（w/w）For 3:1.Each Concentration Testing takes three parallel pipes,
Often pipe adds the μ l of magnetic particle 100 marked with anti-human myoglobins polyclonal antibody, then is separately added into concentration as corresponding concentration
Each 100 μ l of human muscle hemoglobin solution, association reaction shake incubation at 37 DEG C 60 minutes, with magnetic separator adsorbing separation magnetic particle,
Supernatant is abandoned, adds the μ l of PBS 200 cleanings three times, with magnetic separator adsorbing separation magnetic particle, supernatant is abandoned, adds horseradish peroxidase
Enzyme marks the anti-human μ l of myoglobins monoclonal antibody 200, and association reaction shakes incubation at 37 DEG C 60 minutes, is adsorbed with magnetic separator
Magnetic particle is separated, abandons supernatant, the μ l of PBS 200 cleanings is added three times, with magnetic separator adsorbing separation magnetic particle, abandons supernatant, shift
Magnetic particle puts chemiluminescence detector, adds 100 μ l luminous substrate working solutions, when reaction is carried out 2 minutes, record to glow cup
Luminous quantity 6 seconds.
2nd, of the present invention group
The pretreatment of multi-polyester cellulose membrane with experiment one, put seal after 37 DEG C of dryings it is standby.
With experiment one, be placed in preserve in the drying receptacle containing drier makes unclosed group of polyclonal antibody die of the present invention
With.
Inventive closure group polyclonal antibody die carries out the processing with experiment one, and the film produced is put into 37 DEG C of dryings
In case, dry 1 hour.Prepare confining liquid（5% skimmed milk power, 3%BSA, 0.05% tween20,0.8%NaCl, 0.02% KCL,
pH7.4）, the polyclonal antibody die after processing is placed in confining liquid, is stored at room temperature 30 minutes, taking-up is put into 37 DEG C of drying boxes
It is interior, dry 6 hours.
The preparation of chemiluminescent labeling adsorbed film：The multi-polyester cellulose membrane of pretreatment is taken, with 50mM PBSs
(pH 7.4) dilutes the anti-human myoglobins monoclonal antibody 0.15mg/ml of horseradish peroxidase-labeled, starts die instrument, loads
Antibody, takes multi-polyester cellulose membrane, starts die, set die condition as：Airbrush translational speed 30mm/ seconds, liquid promote speed
Spend 3.0 μ l/cm, by after printing film be freeze-dried, put 4 DEG C be sealed it is standby.
Semi-finished product assemble method：Starting dehumidifier makes the humidity in operating room be reduced to less than 25%, is printed in polyclonal antibody
Water suction paper membrane pad and chemiluminescent labeling adsorbed film are pasted in film both ends respectively, then with Pressure sensitive adhesive tape sealing label surface.Put and paste
Detection lug on cutting machine, being cut into 3.5mm test strips.Paper slip is put into the aluminium amber hermetic bag of drier, in sealing machine
Upper sealing, labelling.
The test strips of above-mentioned preparation are taken, with the side of chemiluminescent labeling adsorbed film upward, are placed in centrifuge rotor, to
The human muscle hemoglobin solution 80ul of the various concentrations of preparation is added dropwise on chemiluminescent labeling adsorbed film, stands 3 minutes, 2000 turns/
Separate the heart 1 minute, then the PBS 80ul containing 1% polysorbas20 to dropwise addition pH7.4 on chemiluminescent labeling adsorbed film,
2000 revs/min of centrifugations are cleaned for 1 minute, repeated washing 2 times, are taken out test strips, are cut polyclonal antibody die detection line, be positioned over
In cuvette, PBS 200ul, ultrasonication 3 seconds are added.10ul is taken, Pico luminescence reagents is added, is placed in chemical hair
On optical detector, luminous quantity is kept in reaction in mind when carrying out 2 minutes, and the luminous quantity time of integration is 6 seconds, and experiment in triplicate, as a result takes
Average value, concentration-luminosity curve is then drawn, and calculate coefficient correlation.
3rd, experimental result
The present invention is used to be examined with chemical luminescence detection method and existing chemical luminescence detection method to human muscle hemoglobin solution
Survey, experimental result is as shown in table 4, and as shown in Table 4, good concentration linear relationship, correlation coefficient r is presented in both of which2Value difference
For 0.974 and 0.901, but unclosed group of correlation coefficient r2It is worth for 0.75.Illustrate that inventive closure group has and existing chemistry
The similar Detection results of luminescence technology, but detection time is significantly shorten, unclosed group of background signal is higher, influences to detect.
Table 4 is of the invention with the comparison of the detection performance of existing chemical luminescence detection method（Unit：Luminous value）
| Concentration（ng/ml） | Inventive closure | Prior art | The present invention is unclosed |
| 3.125 | 95380 | 661292 | 1161292 |
| 6.25 | 112336 | 783025 | 1283025 |
| 12.5 | 329378 | 821436 | 1121436 |
| 25 | 956238 | 1123185 | 1323185 |
| 50 | 1608326 | 2234590 | 2420395 |
| 100 | 3082607 | 3411312 | 3518320 |
| r2Value | 0.974 | 0.901 | 0.750 |
Experiment five, the chemiluminescence detection experiment of microballoon of the present invention mediation
Delivery vehicle is reacted by detection of fluorescent microsphere.
First, experiment material
Fluorescent microsphere（The biology of Shanghai outstanding person one）, trehalose（SIGMA）, nitrocellulose filter（Millipore companies）, EDC
（PIERCE companies）, NHS（PIERCE companies）, the anti-human myoglobins monoclonal antibody of horseradish peroxidase-labeled（The U.S.
Genagates companies）, anti-human myoglobins polyclonal antibody（Genagates companies of the U.S.）, spectrophotometer（Shanghai mountain valley with clumps of trees and bamboo China
Tech equipment Co., Ltd, 752 ultraviolet-uisible spectrophotometers）, human muscle hemoglobin（Sigma-Aldrich products）, B
IoFlow die instrument（IMAGENE companies of the U.S.）, Index cutting machines（A-point companies of the U.S.）, DBF-900 sealing machines
（Wenzhou south of the River packing factory）, ACBO dehumidifiers（Jiangsu wuxi Ao Bo dehumidifiers company）, desk centrifuge（U.S. Eppendoff
Company）, bovine serum albumin(BSA)（Abbreviation BSA, SIGMA product）, nitrocellulose diaphragm（AE 99, it is public by U.S. Genagates
Department provides）, multi-polyester cellulose membrane (Reemay 2033, U.S.'s Alstrom Products), absorb water paper membrane pad（Grade
470, U.S.'s S＆S Products）, chemiluminescence detector（Promega, Glomax Multi JR Detection
System）, West Pico luminescence reagents（Thermo scientific）.
2nd, experimental method
The preparation of human muscle hemoglobin solution：With experiment one.
Fluorescent microsphere marks：0.5ml microballoons are taken, using pH6.2 0.1M phosphate buffers eccentric cleaning 4 times,
13000rpm is centrifuged, and is redissolved with pH6.2 0.1M phosphate buffers to 1ml, it is anti-human to add 2mg horseradish peroxidase-labeleds
Myoglobins monoclonal antibody, mix, add 250ul 40mg/ml EDC solution, the NHS for adding 250ul 40mg/ml is molten
Liquid, mix, react at room temperature 60 minutes, add 20mg bovine serum albumin(BSA), mix, react at room temperature 60 minutes.Centrifugation suction is abandoned
Clearly, after using 0.05M Tris pH7.6 eccentric cleanings 4 times, redissolved with 0.5% trehalose, 1%BSA, 0.05M Tris pH7.6
To 10ml, 4 DEG C be kept in dark place it is stand-by.
Fluorescent microsphere mark absorption film preparation：Fluorescent microsphere labelled antibody solution is taken, dilutes 3 times with liquid is redissolved, Qi Tatong
Test the die method in colloid gold mark absorption film preparation.
It is prepared by polyclonal antibody die：With experiment one.
Semi-finished product assemble method：With experiment one.
Experimental group：The test strips of above-mentioned preparation are taken, with the side of fluorescent microsphere mark adsorbed film upward, are placed in centrifugal basket
In son, the human muscle hemoglobin solution 80ul for the various concentrations that preparation is added dropwise on adsorbed film is marked to fluorescent microsphere, stands 3 minutes,
Centrifuged 1 minute with 1000 revs/min, then 0.05% Tween-20,50mM PBS to dropwise addition pH7.4 on fluorescent microsphere mark adsorbed film
Buffer solution 80ul, 2000 revs/min of centrifugations are cleaned for 30 seconds, and cleaning twice, takes out test strips, cuts the detection of polyclonal antibody die
Line, it is positioned in cuvette, adds PBS 200ul, ultrasonication 3 seconds.10ul is taken, Pico luminescence reagents is added, puts
In on chemiluminescence detector, luminous quantity is kept in reaction in mind when carrying out 2 minutes, and the luminous quantity time of integration is 6 seconds, and experiment repeats three
It is secondary, results averaged, concentration-luminosity curve is then drawn, and calculate coefficient correlation.
Control group：The test strips of above-mentioned preparation are taken, are placed in desktop, marks to be added dropwise on adsorbed film to fluorescent microsphere and prepares not
With the human muscle hemoglobin solution 80ul of concentration, stand, treat that the liquid on fluorescent microsphere mark adsorbed film all chromatographs and flow into nitric acid
Cellulose membrane, then 0.05% Tween-20,50mM PBS 80ul to dropwise addition pH7.4 on fluorescent microsphere mark adsorbed film,
Stand, liquid all flows into nitrocellulose filter, and cleaning twice, removes test strips, cuts polyclonal antibody die detection line, put
It is placed in cuvette, adds PBS 200ul, ultrasonication 3 seconds.10ul is taken, adds Pico luminescence reagents, being placed in
Learn on luminometer, luminous quantity is kept in reaction in mind when carrying out 2 minutes, the luminous quantity time of integration is 6 seconds, is tested in triplicate, knot
Fruit is averaged, and then draws concentration-luminosity curve, and calculate coefficient correlation.
3rd, experimental result
This experiment is detected by liquid phase reactor delivery vehicle of fluorescent microsphere, as a result, experimental group test strips single inspection of the present invention
Survey processing time average out to 4.6 minutes, control test group（Do not do centrifugal treating）Test strips single detection process time average out to
49 minutes；Experimental group detection data of the present invention are in the dependency relation of good concentration-luminous value, correlation coefficient r2It is right for 0.987
According to group detection data in below 50ng/ml, the substantially correlation without concentration-luminous value, exist mainly due to chemiluminescent substance
Non-specific binding on immobilon-p can not be effectively cleaned, and cause background too high relevant, correlation coefficient r2For 0.711, P ＜
0.05, result of the present invention is significantly better than the testing result for not doing centrifugal treating, illustrates that the technology of the present invention improves prior art inspection
The linear and accuracy surveyed.Experimental result is as shown in table 5.
The immunochromatography chemiluminescence detection experimental result of the present invention of table 5（Unit：Luminous value）
| Technology | 3.125 | 6.25 | 12.5 | 25 | 50 | 100 | r2 |
| Experimental group | 80235 | 182278 | 387126 | 1232961 | 1782785 | 3672463 | 0.987 |
| Control group | 1261453 | 1288604 | 1322169 | 1297865 | 2321226 | 4120437 | 0.711 |
Experiment six, the of the invention and comparison of existing chemical luminescence detection method testing result
First, experiment material
With experiment five.
2nd, experimental method
Make standard curve：Take concentration known human muscle hemoglobin solution 3.125,6.25,12.5,25,50,100ng/ml people's flesh
Hemoglobin solution, detected using chemical luminescence detection method of the present invention and draw standard curve.With people's flesh red eggs of concentration known
White 30ng/ml is as sample to be checked.Other methods are the same as five experimental groups of experiment.
3rd, experimental result
The concrete outcome for repeating experiment three times is as shown in table 6.Centrifugation technique measurement result of the present invention shows that sample people flesh to be checked is red
The content of albumen is 30.95ng/ml, has good repeatability and accuracy.
The chemical luminescence detection method detection performance experiment of the present invention of table 6（Unit：ng/ml）
| Repeat 1 | Repeat 2 | Repeat 3 | Average value | |
| Centrifugation | 28.76 | 29.55 | 34.55 | 30.95 |
Experiment seven, the of the invention and comparison of existing fluorescence immunoassay detection method detection performance
First, experiment material
Anti-human myoglobins polyclonal antibody（Genagates companies）, anti-human myoglobins monoclonal antibody（The U.S.
Genagates companies）, fluorescent microsphere（Fluorescein used is europium compound, Shanghai one biotech firm of outstanding person）、EDC （Pierce is produced
Product）、NHS（Pierce products）, human muscle hemoglobin（Sigma-Aldrich products）, quantitative fluorescence analysis instrument（Shanghai woman biology
Company, HG-98）, BioFlow die instrument（IMAGENE companies of the U.S.）, Index cutting machines（A-point companies of the U.S.）,
DBF-900 sealing machines（Wenzhou south of the River packing factory）, ACBO dehumidifiers（Jiangsu wuxi Ao Bo dehumidifiers company）, desk-top centrifugation
Machine（Eppendoff companies of the U.S.）, bovine serum albumin(BSA)（SIGMA products）, nitrocellulose diaphragm（AE 99, by the U.S.
Genagates companies provide）, multi-polyester cellulose membrane (Reemay 2033, U.S.'s Alstrom Products), absorb water paper membrane
Pad（Grade 470, U.S.'s S＆S Products）.
2nd, experimental method
The preparation of human muscle hemoglobin solution：With experiment one.
Fluorescent microsphere marks：Take 0.5ml fluorescent microspheres, using PH6.2 0.1M PB eccentric cleanings 4 times, 13000rpm from
The heart, redissolved with pH6.2 0.1M PB to 1ml, add the anti-human myoglobins monoclonal antibodies of 150ug, mixed, add pH6.2's
0.1M PB to 1.5ml, the 250ul 40mg/ml EDC aqueous solution is added, add the 250ul 40mg/ml NHS aqueous solution, mixed
It is even, react at room temperature 60 minutes.20mg BSA is added, is mixed, is reacted at room temperature 60 minutes.Supernatant is abandoned in centrifugation suction, uses 0.05M
After Tris pH7.6 eccentric cleanings 4 times, 4 DEG C preservations stand-by to 10ml are redissolved with 1%BSA, 0.05M Tris pH7.6.
The preparation of fluorescent labeled antibody adsorbed film：Prepare containing 0.5%PVA, 50mM PBS liquid, 0.5%BSA, 0.88%
NaCl, pH 7.4 multi-polyester cellulose membrane pretreatment fluid, pending multi-polyester cellulose membrane is put in pretreatment fluid, room temperature
Immersion 1 hour, takes the film out, put seal after 37 DEG C of dryings it is standby.Fluorescent microsphere labelled antibody solution is taken, with 1%BSA, 0.05M
Tris pH7.6 buffer solutions dilute 3 times, start die instrument, load antibody, take multi-polyester cellulose membrane, start die, setting print
Film condition is：The airbrush translational speed 30mm/ seconds, the μ l/cm of liquid fltting speed 5.0, the film after printing is put into drying box,
37 DEG C of dryings 6 hours, are subsequently placed in preserve in the hermetic bag containing drier and use.
It is prepared by polyclonal antibody die：Anti-human myoglobins Anti-TNF-α liquid solution is taken, with 50mM phosphate buffers
(pH 7.4) is diluted to 1mg/ml concentration.Start die instrument, load antibody, take the PVC pieces for posting nitrocellulose filter, start to print
Film, set die condition as：The airbrush translational speed 30mm/ seconds, the μ l/cm of liquid fltting speed 0.5, the film produced is put into 37
In DEG C drying box, dry 6 hours, then be placed in preserve film in the drying receptacle containing drier and use.
Semi-finished product assemble method：Starting dehumidifier makes the humidity in operating room be reduced to less than 25%, is printed in polyclonal antibody
Water suction paper membrane pad and fluorescent labeled antibody adsorbed film are pasted in film both ends respectively, then with Pressure sensitive adhesive tape sealing label surface.Put and paste
Detection lug on cutting machine, being cut into 3.5mm test strips.Paper slip is put into the aluminium amber hermetic bag of drier, in sealing machine
Upper sealing, labelling.
The test strips of above-mentioned preparation are taken, with the side of fluorescent labeled antibody adsorbed film upward, are placed in centrifuge rotor, to
The human muscle hemoglobin solution 80ul of the various concentrations of preparation is added dropwise on fluorescent labeled antibody adsorbed film, stands 3 minutes, 2000 turns/
Separate the heart 1 minute, then the PBS 80ul to dropwise addition pH7.4 on fluorescent labeled antibody adsorbed film, 2000 revs/min centrifuge 1 point
Clock is cleaned, and takes out test strips, and the fluorescent value of polyclonal antibody trace band is read on quantitative fluorescence analysis instrument.
Current art control stripes bar does not do centrifugal treating, after 3 minutes time of repose of setting, then stands 2.5 minutes,
Then fluorescent value is read.
Test in triplicate, results averaged.
3rd, experimental result
Concrete outcome is as shown in table 7.For the technology of the present invention through operating as above, linear response is good, correlation coefficient r2It is worth for 0.994.
Determined using prior art, 3 points are stood after point sample, then stand 2.5 points, linear bad, below 12.5ng/ml sample, it is sent out
Light quantity is close to background level, correlation coefficient r2It is worth for 0.900.
Table 7 is of the invention with the comparison of the detection performance of existing immunofluorescent detection method（Unit：Luminous value）
| Concentration（ng/ml） | The present invention | Prior art |
| 3.125 | 18820 | 84125 |
| 6.25 | 45323 | 87873 |
| 12.5 | 97832 | 92451 |
| 25 | 152371 | 186782 |
| 50 | 324569 | 317653 |
| 100 | 632872 | 587749 |
| r2 | 0.994 | 0.900 |
Experiment eight, the of the invention and comparison of existing immunofluorescent detection method testing result
First, experiment material
Sheep anti-mouse igg polyclonal antibody（Genagates companies of the U.S. provide）, it is other with experiment seven.
2nd, experimental method
The preparation of human muscle hemoglobin solution：With experiment one.Prepare the human muscle hemoglobin measuring samples of 20ng/ml concentration knowns.
Make standard curve：Take concentration known human muscle hemoglobin solution 3.125,6.25,12.5,25,50,100ng/ml
Human muscle hemoglobin solution, is respectively adopted the present invention and current art detects and draws standard curve.
Fluorescent microsphere marks：With experiment seven.
The printing of fluorescent microsphere film：With experiment seven.
The preparation of fluorescent labeled antibody adsorbed film：With experiment seven.
It is prepared by polyclonal antibody die：Anti-human myoglobins Anti-TNF-α liquid solution is taken, with 50mM phosphate buffers
(pH 7.4) is diluted to 1mg/ml concentration.Sheep anti-mouse igg Anti-TNF-α liquid solution is taken, with 50mM phosphate buffers (pH 7.4)
It is diluted to 1mg/ml concentration.Start die instrument, load antibody, take the PVC pieces for posting nitrocellulose filter, start die, it is same
Anti-human myoglobins polyclonal antibody is printed on nitrocellulose filter as detection line T, sheep anti-mouse igg polyclonal antibody as matter
Control line C, set die condition as：The airbrush translational speed 30mm/ seconds, the μ l/cm of liquid fltting speed 0.5, the film produced is put
Enter in 37 DEG C of drying boxes, dry 6 hours, then film is placed in the drying receptacle containing drier and preserves use.
Semi-finished product assemble method：With experiment seven.
The test strips of above-mentioned preparation are taken, with the side of fluorescent labeled antibody adsorbed film upward, are placed in centrifuge rotor, to
The human muscle hemoglobin solution and each 80ul of testing sample of the various concentrations of preparation are added dropwise on fluorescent labeled antibody adsorbed film, stands 3
Minute, 2000 revs/min centrifuge 1 minute, then to the PBS 80ul that pH7.4 is added dropwise on fluorescent labeled antibody adsorbed film, 2000
Rev/min centrifugation 1 minute clean, take out test strips, on quantitative fluorescence analysis instrument read polyclonal antibody printed film on detection line T
With nature controlling line C fluorescent value, and T/C ratios are calculated, draw standard curve, calculate measuring samples myoglobin concentration.
Current art control stripes bar does not do centrifugal treating, after the time of repose of setting, then stands 2.5 minutes, then
Fluorescent value is read, and calculates T/C ratios, draws standard curve, calculates measuring samples myoglobin concentration.
Test in triplicate, results averaged.
3rd, experimental result
For the technology of the present invention through operating as above, standard curve is linearly good, correlation coefficient r2It is worth for 0.995, has carried out sample immediately
Measure, the ng/ml of empirical average 19.12, meets the requirements three times.Determined using prior art, 3 points are stood after point sample, then stand
2.5 points, examination criteria curve linear is bad, correlation coefficient r2It is worth for 0.937.Then time of repose after total point sample is extended to
15 minutes of existing Product checking, standard curve is linearly good, correlation coefficient r2It is worth for 0.961, immediately according to the bar of 15 minutes
Part has carried out sample measure, and average value is 20.84ng/ml three times, is met the requirements.The concrete outcome such as table 8 of experiment is repeated three times
It is shown.Compared with the prior art, the present invention significantly shorten detection time.
The present invention of table 8 is with the Analysis of test results of immunofluorescent detection method（Unit：ng/ml）
| Repeat 1 | Repeat 2 | Repeat 3 | Average value | |
| The present invention | 16.98 | 21.22 | 19.17 | 19.12 |
| Prior art | 22.35 | 21.21 | 18.97 | 20.84 |
Experiment nine, fluoroscopic examination of the present invention and the comparison of existing enzyme-linked immune detection method testing result
First, experiment material
Enzyme-linked immunosorbent assay instrument（Bio-Rad, Model 550）, Healthy Human Serum（Healthy premenopausal volunteers are donated）, it is other with experiment
Seven.
2nd, experimental method
The preparation of human muscle hemoglobin solution：With experiment one.
Measuring samples are used as using the 16.1ng/ml human muscle hemoglobins Healthy Human Serum of concentration known.
Make standard curve：Take concentration known human muscle hemoglobin solution 3.125,6.25,12.5,25,50,100ng/ml
Human muscle hemoglobin solution, fluoroscopic examination of the present invention and existing enzyme-linked immune detection method detection human muscle hemoglobin solution is respectively adopted
And standard curve is drawn, then take measuring samples to determine, the concentration of myoglobins in measuring samples is calculated with standard curve.Each
Sample makees 3 parallel pipes.
1st, existing enzyme-linked immune detection method group
Using 96 hole elisa plates, the anti-human μ l of myoglobins polyclonal antibody 100 of often pipe addition, 4 DEG C are coated with overnight, clean three times,
Human muscle hemoglobin solution or the μ l of measuring samples 100 are separately added into again, and association reaction incubates 120 minutes at 37 DEG C, and cleaning three times, adds
Enter the anti-human μ l of myoglobins monoclonal antibody 100, association reaction incubates 60 minutes at 37 DEG C, and cleaning three times, abandons supernatant, adds peppery
The μ l of root peroxidase labelling sheep anti-mouse igg polyclonal antibody 100, association reaction are incubated 60 minutes at 37 DEG C, and cleaning three times, is abandoned
Supernatant, add 100 μ l nitrite ions（Formula：0.1M citric acids 2.43ml, 0.2M disodium hydrogen phosphate 2.57ml, o-phenylenediamine 5mg,
The μ l of hydrogen peroxide 5）, lucifuge 5 minutes, add 2M sulfuric acid terminating reactions.Reading OD490 light absorption values on enzyme-linked immunosorbent assay instrument are put,
Draw standard curve, r2=0.991, calculate human muscle hemoglobin content in measuring samples.
2nd, of the present invention group
Fluorescent microsphere marks：With experiment seven.
The printing of fluorescent microsphere film：With experiment seven.
The preparation of fluorescent labeled antibody adsorbed film：With experiment seven.
It is prepared by polyclonal antibody die：With experiment eight.
Semi-finished product assemble method：With experiment seven.
The test strips of above-mentioned preparation are taken, with the side of fluorescent labeled antibody adsorbed film upward, are placed in centrifuge rotor, to
The human muscle hemoglobin solution and each 80ul of testing sample of the various concentrations of preparation are added dropwise on fluorescent labeled antibody adsorbed film, stands 3
Minute, 2000 revs/min centrifuge 1 minute, then to the PBS 80ul that pH7.4 is added dropwise on fluorescent labeled antibody adsorbed film, 2000
Rev/min centrifugation 1 minute clean, take out test strips, on quantitative fluorescence analysis instrument read polyclonal antibody print printed film on detects
Line T and nature controlling line C fluorescent value, and T/C ratios are calculated, draw standard curve, r2=0.987, calculate measuring samples myoglobins
Concentration.
3rd, experimental result
Concrete outcome is as shown in table 9.Existing enzyme-linked immune detection method measurement result shows containing for sample human muscle hemoglobin to be checked
Measure as 16.54ng/ml, measurement result of the present invention shows that the content of sample human muscle hemoglobin to be checked is 16.91ng/ml, two kinds of realities
Proved recipe method acquired results are basically identical, no difference of science of statistics（P>0.05）, but the completion experimental period of the present invention is significantly shorter than now
Row technology.
9 fluoroscopic examination of the present invention of table and the comparison of existing enzyme-linked immune detection method testing result（Unit：ng/ml）
| Repeat 1 | Repeat 2 | Repeat 3 | Average value | |
| Current art | 18.72 | 17.28 | 13.63 | 16.54 |
| The present invention | 15.44 | 18.91 | 16.37 | 16.91 |
Experiment ten, influence of the centrifugal speed of the present invention to testing result
First, experiment material
With experiment 1
2nd, experimental method
With experiment 1, pre-dwell 3 minutes.
3rd, experimental result
The present invention, using different centrifugal speeds, have detected the human muscle hemoglobin sample of various concentrations using collaurum as indicator, real
It is as shown in table 10 to test result.As shown in Table 10, detection accuracy is relevant with centrifugal speed, and 500,1000,2000,3000,4000
Rev/min centrifugal speed obtain satisfactory testing result, correlation coefficient r value is all higher than 0.98.4000th, 5000 revs/min
The testing result that centrifugal speed obtains, its correlation coefficient r value are below 0.98, are not reaching to coherent detection requirement.Illustrate this hair
The bright optimal centrifugal speed for carrying out myoglobins detection should be below 3000 revs/min.
Influence of the centrifugal speed of table 10 to testing result
| Centrifugal speed | 3.125 | 6.25 | 12.5 | 25 | 50 | 100 | r2 |
| 500r/min | 30.2 | 34.7 | 40.7 | 49.2 | 63.8 | 78.5 | 0.988 |
| 1000r/min | 29.7 | 32.0 | 38.9 | 50.6 | 61.8 | 79.8 | 0.980 |
| 2000r/min | 28.8 | 33.5 | 38.0 | 46.8 | 56.4 | 82.4 | 0.962 |
| 3000r/min | 28.1 | 33.2 | 37.7 | 49.2 | 56.4 | 74.3 | 0.987 |
| 4000r/min | 19.2 | 24.8 | 26.6 | 28.4 | 34.7 | 66.9 | 0.835 |
| 5000r/min | 19.8 | 21.5 | 26.4 | 28.1 | 34.2 | 62.0 | 0.864 |
Test 11, influence of the diameter of particle of the present invention to testing result
Using polystyrene microsphere as liquid phase reactor delivery vehicle.
First, experiment material
Polystyrene microsphere（Particle diameter 35,130,376,600,1000nm, carboxylated, upper great waves space are international）, it is other with experiment one.
2nd, experimental method
The preparation of human muscle hemoglobin solution：With experiment one.
Anti-human myoglobins monoclonal antibody FITC marks：Using Marsshall methods, the anti-human myoglobins lists of 3mg/ml are taken
Clonal antibody solution, add the 0.5M of 1/10 volume（pH9.0）Carbonate buffer solution, electromagnetic agitation 5 minutes.With 50mM phosphate
（PBS）, pH8.0 buffers concentration is 1mg/ml FITC solution, is slowly added to 30ul/ml amount under stirring
In antibody-solutions, 4 DEG C of lucifuges stir 12 hours.The antibody-solutions of mark are centrifuged（2500r/min）20 minutes, remove precipitation
Thing, it is fitted into bag filter, is placed in 4 DEG C of dialysed overnights of 50mM PBSs.The label of dialysed overnight is taken, crosses sephadex
G-25 posts, free FITC is separated, collect the fluorescence antibody of mark, be i.e. FITC marks anti-human myoglobins monoclonal antibody, -20 DEG C
It is kept in dark place standby.
Polystyrene microsphere marks：2ml 0.1M MES, pH5.0 solution, FITC is taken to mark anti-human myoglobins monoclonal
Antibody 2mg, 0.2ml 5% w/v microballoons and 20mg EDC, mix, add 10mg NHS, vibrated at room temperature in impeller
Placed 2 hours on device, 12000rpm is centrifuged 15 minutes, is abandoned supernatant, is added 2ml 20mM PBS, 1%BSA Block buffer room temperature
Closing 1 hour, 4ml 50mM PBS, pH7.4 buffer solution is added to be suspended, repeated washing once, adds 2ml 50mM PBSs,
4 DEG C save backup.
Polystyrene microsphere mark absorption film preparation：Polystyrene microsphere labelled antibody solution is taken, with 50mM PBS,
PH7.4 buffer solutions are diluted to 1% w/v microballoons, start die instrument, load antibody, open pressurized nitrogen, take poly ester fiber element
Film, start die, set die condition as：Airbrush translational speed 30mm/ seconds, the μ l/cm of liquid fltting speed 5.0, after printing
Film be put into drying box, 37 DEG C of dryings 6 hours are subsequently placed in preserve in the hermetic bag containing drier and used.
It is prepared by polyclonal antibody die：With experiment one.
Semi-finished product assemble method：With experiment one.
Experimental group：The test strips of above-mentioned preparation are taken, with the side of polystyrene microsphere mark adsorbed film upward, are placed in centrifugation
In machine rotor, to polystyrene microsphere mark adsorbed film on be added dropwise preparation various concentrations human muscle hemoglobin solution 80ul, it is quiet
Put 3 minutes, centrifuged 1 minute with 1000 revs/min, then 0.05% tween to dropwise addition pH7.4 on polystyrene microsphere mark adsorbed film
20th, 50mM PBSs 80ul, 2000 revs/min of centrifugations are cleaned, take out test strips, be positioned on fluorescence detector and read for 30 seconds
Fluorescent value, experiment in triplicate, results averaged, then draw concentration-luminosity curve, and calculate coefficient correlation.
3rd, experimental result
The present invention is using polystyrene microsphere as liquid phase reactor delivery vehicle, influence of the observation different-grain diameter to experimental result.As a result,
Particle diameter of the present invention is 35,130,376,600,1000nm detection data be in good concentration-luminous value dependency relation, correlation
Coefficient r is all higher than 0.98, illustrates that the particulate of different-grain diameter is applied to the technology of the present invention.Experimental result is as shown in table 11.
Influence of 11 diameter of particle of the present invention of table to testing result（Unit：Luminous value）
| Particle diameter（nm) | 3.125 | 6.25 | 12.5 | 25 | 50 | 100 | r2 |
| 35 | 83160 | 113520 | 225362 | 285321 | 391786 | 450012 | 0.955 |
| 130 | 95461 | 168213 | 213382 | 242914 | 322769 | 394864 | 0.950 |
| 376 | 109234 | 156378 | 223451 | 267869 | 297712 | 403524 | 0.967 |
| 600 | 94532 | 134568 | 212219 | 278763 | 318826 | 398772 | 0.964 |
| 1000 | 95674 | 112367 | 198756 | 287964 | 312278 | 412344 | 0.956 |
Test 12, influence of the cleaning step of the present invention to testing result
First, experiment material
With experiment one.
2nd, experimental method
Control experiment cleaning step is stood, and is not cleaned.Other same experiments one.
3rd, experimental result
Experimental result is as shown in table 12.Two test results compare, and the testing result of cleaning is better than not cleaning, correlation coefficient r2Value
Respectively 0.968 contains 0.943.Illustrate test sample introduction after with cleaning the step of be necessary.
Influence of 12 cleaning step of the present invention of table to testing result（Unit：Pattern colour angle value）
| Concentration（ng/ml） | Cleaning | It is no clean |
| 3.125 | 20.3 | 29.6 |
| 6.25 | 22.2 | 28.7 |
| 12.5 | 24.3 | 33.5 |
| 25 | 31.1 | 37.1 |
| 50 | 38.6 | 41.4 |
| 100 | 47.7 | 48.6 |
| r2 | 0.968 | 0.943 |
Test 13, influence of the Avidin biotin amplification system to detection performance of the present invention
Using colloidal gold particles as liquid phase reactor delivery vehicle.
First, experiment material
NHS activated biotins（SIGMA）, Avidin（SIGMA）, it is other with experiment one.
2nd, experimental method
The preparation of human muscle hemoglobin solution：With experiment one, concentration 0.1,1,3.13,6.25,12.5,25,50,100ng/ml.
Anti-human myoglobins polyclonal antibody biotin labeling：Anti-human myoglobins polyclonal antibody is taken in 0.1M pH9.5
Dialysed overnight in sodium carbonate buffer, adjust final concentration of 2mg/ml.NHS activated biotins 20mg is taken to be dissolved in 1ml dimethyl methyls
Acid amides, 50ul is taken, be added in above-mentioned solution, reacted at room temperature 4 hours.Reaction solution is dialyzed overnight in PBS, -20 DEG C
Preserve.
Colloidal gold particles mark：With experiment one.
Colloidal gold particles mark absorption film preparation：Colloidal gold particles labelled antibody solution is taken, with colloid buffer solution（1%BSA,
3% sucrose, 50mM PBS, pH7.4）It is 30 to be diluted to OD530, adds the anti-human myoglobins polyclonal antibody 10 of biotin labeling
μ g/ml, mix.Other same experiments one.
It is prepared by Avidin die：Avidin solution is taken, it is dense to be diluted to 1mg/ml with 50mM phosphate buffers (pH 7.4)
Degree.Start die instrument, load avidin solution, it is other with experiment one.
Semi-finished product assemble method：Starting dehumidifier makes the humidity in operating room be reduced to less than 25%, in Avidin die two
Water suction paper membrane pad and colloidal gold particles mark adsorbed film are pasted in end respectively, other with experiment one.
Experimental group：The test strips of above-mentioned preparation are taken, with the side of colloidal gold particles mark adsorbed film upward, are placed in centrifuge
In rotor, the human muscle hemoglobin solution 80ul for the various concentrations that preparation is added dropwise on adsorbed film is marked to colloidal gold particles, stands 3 points
Clock, with 1000 revs/min centrifuge 1 minute, then to colloidal gold particles mark adsorbed film on be added dropwise pH7.4 0.05% Tween-20,
50mM PBSs 80ul, 2000 revs/min of centrifugations are cleaned for 30 seconds, take out test strips, are positioned over the quantitative chromatographic analysis of collaurum
Chromatic value is read on instrument, experiment in triplicate, results averaged, then draws concentration-chromatic value curve, and calculate phase relation
Number.
Control group：The test strips of above-mentioned preparation are taken, are placed in desktop, is marked to colloidal gold particles and is added dropwise what is prepared on adsorbed film
The human muscle hemoglobin solution 80ul of various concentrations, stand, treat the red colloid gold particulate mark on colloidal gold particles mark adsorbed film
Remember thing all chromatography flow into nitrocellulose filter, then to colloidal gold particles mark adsorbed film on be added dropwise pH7.4 0.05% tween-
20th, 50mM PBSs 80ul, stand, liquid all flows into nitrocellulose filter, removes test strips, is positioned over collaurum and determines
Chromatic value is read on amount chromatographic analysis instrument, experiment in triplicate, results averaged, then draws concentration-chromatic value curve, and
Calculate coefficient correlation.
3rd, experimental result
The present invention is using colloidal gold particles as liquid phase reactor delivery vehicle, as a result, experimental group test strips single detection process of the present invention
Time average out to 3.7 minutes, control test group（Do not do centrifugal treating）Test strips single detection process time average out to 38 minutes；
Experimental group detection data of the present invention are in the dependency relation of good concentration-chromatic value, correlation coefficient r2For 0.973, linearity test
Scope is 0.1-100 ng/ml；The correlation r of control group detection2For 0.902, not up to r is more than 0.98 testing requirements, but
When linear detection range is adjusted into 3.125-100 ng/ml, correlation coefficient r2For 0.973, the inspection that r is more than 0.98 is reached
Survey and require.Experimental result illustrates that the technology of the present invention not only shortens detection time, while improves the sensitive of prior art detection
Degree and accuracy.Experimental result is as shown in table 13.
Influence of the Avidin biotin amplification system of table 13 to detection performance of the present invention（Unit：Chromatic value）
| Concentration（ng/ml） | Amplification | Do not amplify |
| 0.1 | 10.2 | 12.6 |
| 1 | 16.4 | 13.8 |
| 3.125 | 18.6 | 16.8 |
| 6.25 | 22.8 | 24.5 |
| 12.5 | 24.5 | 35.0 |
| 25 | 29.7 | 38.6 |
| 50 | 40.2 | 48.2 |
| 100 | 46.8 | 56.4 |
| r2 | 0.973 | 0.902 |
Claims (10)
1. one kind centrifuges detection method, methods described is to be detected using centrifugal device driving liquid phase in immunochromatography solid phase
Flowed in film, wherein the immunochromatography include using colloidal metal as indicator colloidal metal immunochromatography, using fluorescein as
The luminous chemiluminescence as indicator of fluorescence immune chromatography and chemiluminescent substance and/or chemiluminescence enzyme mediation of indicator
At least one of immunochromatography.
2. method according to claim 1, it is characterised in that：The colloidal metal is collaurum, electroselenium and collaurum magnetic
At least one of particulate；The fluorescein includes fluorescein isothiocynate, RB 200, tetramethyl isothiocyanate Luo Dan
Bright, phycoerythrin, perdinin phyllochlorin, propidium iodide, other at least one of phycocyanin and europium compound；Institute
Stating chemiluminescent substance includes luminol and different luminol and its derivative species, acridinium ester and a word used for translation shallow lake amide-type, (golden steel alkane) -1,
2- dichloroethanes and its at least one of derivative and tris (bipyridine) ruthenium；The chemiluminescence enzyme include HRPO,
At least one of alkaline phosphatase and xanthine oxidase.
3. method according to claim 1, it is characterised in that：Solid phase detection film include nitrocellulose filter, pvdf membrane,
Polyvinylidene fluoride film, nylon membrane, one kind of DEAE cellulose membranes or combination.
4. method according to claim 1, it is characterised in that：Methods described is that the carrying using particulate as the indicator carries
Body；The mode that the carrying carrier carries the indicator uses the thing to be checked specificity directly marked with the indicator direct
Conjugate or thing specificity indirect conjugates to be checked are marked the particulate or directly marked with the particulate with the indicator
Remember thing specificity to be checked directly in conjunction with thing or thing specificity indirect conjugates to be checked；The particulate is directly and/or can pass through
Chemical crosslinking mode forms non-specific binding with protein and/or the indicator and maintains stable particle, the particulate
Particle diameter selection 1nm-1um；The specificity junction mixture includes antigen, antibody, Avidin, biotin and its derivative.
5. method according to claim 1, it is characterised in that：The liquid phase includes the liquid phase containing thing to be checked, with the finger
Show the liquid phase for the detectable substance that agent marks, the liquid phase of the detectable substance marked with the particulate, the liquid phase of the detectable substance of non-marked, cleaning
One kind of liquid phase or combination；The detectable substance is thing specificity junction mixture to be checked and its two level or three-level specificity junction mixture, institute
Stating specificity junction mixture includes antigen, antibody, Avidin, biotin and its derivative.
6. method according to claim 1, it is characterised in that：Methods described is also containing solid phase detection membrane closure liquid, institute
State confining liquid be containing bovine serum albumin(BSA) and other soluble proteins, skimmed milk power, polyethylene glycol, casein, sucrose,
The buffer salt solution of at least one of surfactant, gelatin, serum, blood plasma, including step used below：
1）Film coating buffer is detected using solid phase and is coated with the solid phase detection film, the solid phase detection film coating buffer includes antigen, resisted
Body, Avidin, the solution of biotin and its derivative；
2）The solid phase detection film is cleaned using the cleaning fluid, the cleaning fluid includes water, conventional buffers and its contains table
The cushioning liquid of face activating agent；
3）Oozed with confining liquid leaching and cover the solid phase detection film；
4）The solid phase detection film is cleaned again with the cleaning fluid；
5）Dry, it is standby.
7. one kind, which centrifuges detection means, includes sample introduction part and centrifugal device；The centrifugal device includes being driven by motor
Dynamic centrifugal rotor and support base, the centrifugal rotor is using the support base as support；The sample introduction part not with centrifugation
Rotor is directly connected to, located at the top of centrifugal rotor, lower section or outside；The sample introduction part includes liquid phase storage device, sample introduction
Pipe and sampling pump；The liquid phase storage device is connected with the sample feeding pipe；The sampling pump drives the liquid phase storage device
In liquid enter the sample feeding pipe；The sample feeding pipe directly or indirectly loads the liquid phase to the nearly heart of solid phase detection film
On side；The solid phase detection film, which is placed on the centrifugal rotor, to be centrifuged.
8. device according to claim 7, it is characterised in that：Described device contains detector, including collaurum quantitatively detects
Device, fluorescence detector, one kind of chemiluminescence detector or combination, the Testing index of the detector include absorbance, fluorescence
Any of value, values of chemiluminescence and image digital signal value or combination.
9. according to the described device of claim 7 or 8, it is characterised in that：The rotating speed of the centrifugal device and the sampling device
Sample introduction speed uses program controlled mode；The rotating speed of the centrifugal device is 200 ~ 10000 r/min.
10. the application of method or described device in immunoassay technology product development any one of claim 1-9.
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| CN201710212197.9A CN107525923A (en) | 2017-04-01 | 2017-04-01 | One kind centrifuges immunochromatography detection method and device |
| PCT/CN2018/088394 WO2018177445A1 (en) | 2017-04-01 | 2018-05-25 | Centrifugation immunochromatography detection method and apparatus |
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| CN110646616A (en) * | 2019-09-05 | 2020-01-03 | 桂林理工大学 | Hypersensitivity fluorescence quenching immunosensor for detecting human cTnI in serum and detection method |
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